1. Field of the Invention
The field of the invention is odorant receptors.
2. Background of the Invention
The detection and discrimination of the multitude of environmental stimuli by the vertebrate olfactory system results from the activation of olfactory neurons within the olfactory epithelium of the nose (reviewed by Shepherd, 1994; Buck, 1996). The first step in olfactory processing resides at the level of the interaction of odorous ligands with odorant receptors. A large multigene family thought to encode odorant receptors was initially identified in the rat (Buck and Axel, 1991). These receptors are predicted to exhibit a seven transmembrane domain topology characteristic of the superfamily of G protein-coupled receptors. The sizes of the receptor repertoires of different vertebrate species are extremely large and are estimated to contain between 100 and 1000 individual genes (Buck, 1996). These observations suggest that the initial step in olfactory discrimination is accomplished by the integration of signals from a large number of specific receptors, each capable of binding only a small number of structurally-related odorants. Consistent with this model, it has been shown that one rat odorant receptor can be activated by 7 to 10 carbon n-aliphatic aldehydes (Zhao et al., 1997; see also Krautwurst et al., 1998; Malnic et al., 1999). In invertebrates, the C. elegans odr-10 gene encodes a G protein-coupled receptor that is sharply tuned to respond to the odorant, diacetyl (Sengupta et al., 1996; Zhang et al., 1997).
Other olfactory G protein-coupled receptors unrelated to the receptor gene family first described by Buck and Axel (1991) have been identified in the vomeronasal organ (VNO) of mammals (Dulac and Axel, 1995; Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997). The VNO is a specialization of the peripheral olfactory system in higher vertebrates that receives non-volatile pheromonal and non-pheromonal cues (Halpern, 1987). The VNO receptors are encoded by two unrelated gene families; members of the VNR family are localized in a subpopulation of VNO neurons defined by their expression of the G protein alpha subunit, Gai2 (Dulac and Axel, 1995; Berghard and Buck, 1996; Jia and Halpern, 1996), whereas members of the V2R family are expressed predominantly in a separate subpopulation of Gao-expressing cells (Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997). Interestingly, the V2R receptors are structurally related to the calcium-sensing receptor (CaSR; Hebert and Brown, 1995) and metabotropic glutamate receptor (mGluR; Tanabe et al., 1992) families. While it has been proposed that both classes of VNO receptors comprise pheromone receptors, the actual function of these orphan receptors awaits a direct demonstration of their ligand binding or ligand activation properties.
As an approach toward identifying ligands for olfactory receptors, we have pursued an expression cloning strategy using the goldfish as a model system. Fish are thought to respond to a smaller range of odorants than terrestrial vertebrates and thus appear to possess a smaller repertoire of odorant receptors (Ngai et al., 1993b). Moreover, the odorants that fish detect are water soluble, and include amino acids (feeding cues), bile acids (nonreproductive social cues with possible roles in migration), and sex steroids and prostaglandins (sex pheromonal cues) (reviewed by Hara, 1994; Sorensen and Caprio, 1998). Electrophysiological studies have defined the sensitivities of fish olfactory systems to specific ligands, demonstrating, for example, thresholds for detection in the picomolar (for sex steroids) to nanomolar (for amino acids) range (Hara, 1994). Thus, the defined properties of odorant-evoked pathways in vivo provide an excellent starting point for the molecular and biochemical characterization of fish odorant receptors.
In the examples below, we describe the expression cloning of a cDNA encoding a goldfish odorant receptor preferentially tuned to recognize basic amino acids. This cDNA encodes a G protein-coupled receptor that shares significant similarity to receptor families that include the CaSR, mGluR, and V2R class of VNO receptors. Degenerate polymerase chain reaction (PCR) reveals other related sequences that are expressed in the goldfish olfactory epithelium. Together our results indicate that these receptors comprise a family of odorant receptors. Moreover, the characterization of the goldfish amino acid receptor""s odorant tuning properties provides critical molecular parameters for considering models of molecular recognition and information coding in the olfactory system.
Aspects of this invention have been published by Speca et al. (Neuron 1999 July; 23(3):487-98).
The invention provides methods and compositions relating to odorant receptors, including a general expression cloning methodology which is useful for identifying novel G protein-coupled receptors and a novel family of odorant receptors and related nucleic acids, ligands, agonists and antagonists. These compositions provide a wide variety of applications such as screening for related receptors, and by modulating the function of the disclosed receptors by modulating their expression or contacting them with agonists, antagonist or ligands modulating reproductive/sexual and non-sexual social behaviors mediated via the olfactory system, reproductive physiologies and olfactory system regulated feeding behaviors, migratory behaviors and events such as conception, implantation, estrous and menstruation.
The following description of particular embodiments and examples are offered by way of illustration and not by way of limitation. While particularly directed and exemplified often in terms of goldfish R5.24, the following descriptions, including fragment limitations and assay utilizations, also apply to the other disclosed CaSR-like polypeptides and polynucleotides.
The subject domains provide R5.24 domain specific activity or function, such as R5.24-mediated olfaction, ligand signal transducing or transducing inhibitory activity and/or R5.24-specific binding target-binding or binding inhibitory activity. R5.24-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals (e.g. gene therapy, transgenics, etc.), etc. The specific binding target may be a ligand, agonist or antagonist, a R5.24 regulating protein or other regulator that directly modulates R5.24 activity or its localization; or non-natural binding target such as a specific immune protein such as an antibody, or a R5.24 specific agent such as those identified in screening assays such as described below. R5.24-binding specificity may be assayed by binding equilibrium constants (usually at least about 107 Mxe2x88x921, preferably at least about 108 Mxe2x88x921, more preferably at least about 109 Mxe2x88x921), by the ability of the subject polypeptides to function as negative mutants in R5.24-expressing cells, to elicit R5.24 specific antibody in a heterologous host (e.g. a rodent or rabbit), etc.
Exemplary suitable R5.24 polypeptides (a) SEQ ID NO:02, or a functional deletion mutant thereof or a sequence about 60-70%, preferably about 70-80%, more preferably about 80-90%, more preferably about 90-95%, most preferably about 95-99% similar to the R5.24 sequence disclosed herein as determined by Best Fit analysis using default settings and/or (b) is encoded by a nucleic acid comprising a natural R5.24 encoding sequence (such as SEQ ID NO:01) or a fragment thereof at least 36, preferably at least 72, more preferably at least 144, most preferably at least 288 nucleotides in length which specifically hybridizes thereto. Suitable deletion mutants are readily screened in R5.24 binding or activation assays as described herein. Preferred R5.24 domains/deletion mutants/fragments comprise at least 8, preferably at least 16, more preferably at least 32, most preferably at least 64 consecutive residues of SEQ ID NO:2 and provide a R5.24 specific activity, such as R5.24-specific antigenicity and/or immunogenicity, especially when coupled to carrier proteins. The subject domains provide R5.24-specific antigens and/or immunogens, especially when coupled to carrier proteins. For example, peptides corresponding to R5.24-specific domains are covalently coupled to keyhole limpet antigen (KLH) and the conjugate is emulsified in Freunds complete adjuvant. Laboratory rabbits are immunized according to conventional protocol and bled. The presence of R5.24-specific antibodies is assayed by solid phase immunosorbant assays using immobilized R5.24 polypeptides. R5.24 specific antigenic and/or immunogenic peptides encompass diverged sequence regions, preferably diverged extracellular or cytosolic regions, as seen in alignments with related sequences human CaSR, Fugu Ca02.1, mouse V2R2 and rat mGluR1.
Suitable natural R5.24 encoding sequence fragments are of length sufficient to encode such R5.24 domains. In a particular embodiment, the R5.24 fragments comprise species specific fragments; such fragments are readily discerned from alignments. Exemplary such R5.24-1 immunogenic and/or antigenic peptides are shown in Table 1.
In one embodiment, the R5.24 polypeptides are encoded by a nucleic acid comprising SEQ ID NO:01 or a fragment thereof which hybridizes with a full-length strand thereof, preferably under stringent conditions. Such nucleic acids comprise at least 36, preferably at least 72, more preferably at least 144 and most preferably at least 288 nucleotides of SEQ ID NO:01. Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5xc3x97SSPE (0.18 M NaCl, 0.01 M NaPO4, pH7.7, 0.001 M EDTA) buffer at a temperature of 42xc2x0 C. and remaining bound when subject to washing at 42xc2x0 C. with 0.2xc3x97SSPE (Conditions I); preferably hybridizing in a buffer comprising 50% formamide in 5xc3x97SSPE buffer at a temperature of 42xc2x0 C. and remaining bound when subject to washing at 42xc2x0 C. with 0.2xc3x97SSPE buffer at 42xc2x0 C. (Conditions II). Exemplary nucleic acids which hybridize with a strand of SEQ ID NO:01 are shown in Table 2.
A wide variety of cell types express R5.24 polypeptides subject to regulation by the disclosed methods, including many neuronal cells, transformed cells, infected (e.g. virus) cells, etc. Ascertaining R5.24 binding or activation is readily effected by binding assays or cells function assays as disclosed herein. Accordingly, indications for the subject methods encompass a wide variety of cell types and function, etc. The target cell may reside in culture or in situ, i.e. within the natural host.
In another aspect, the invention provides methods of screening for agents which modulate R5.24-ligand interactions. These methods generally involve forming a mixture of a R5.24-expressing cell, a R5.24 ligand and a candidate agent, and determining the effect of the agent on the R5.24-ligand interaction. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.
The amino acid sequences of the disclosed R5.24 polypeptides are used to back-translate R5.24 polypeptide-encoding nucleic acids optimized for selected expression systems (Holler et al. (1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166) or used to generate degenerate oligonucleotide primers and probes for use in the isolation of natural R5.24-encoding nucleic acid sequences (xe2x80x9cGCGxe2x80x9d software, Genetics Computer Group, Inc, Madison Wis.). R5.24-encoding nucleic acids are used in R5.24-expression vectors and incorporated into recombinant host cells, e.g. for expression and screening, etc.
The invention also provides nucleic acid hybridization probes and replication/amplification primers having a R5.24 cDNA specific sequence comprising a fragment of SEQ ID NO:1, and sufficient to effect specific hybridization thereto. Such primers or probes are at least 12, preferably at least 24, more preferably at least 36 and most preferably at least 96 nucleotides in length. Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5xc3x97SSPE (0.18 M NaCl, 0.01 M NaPO4, pH7.7, 0.001 M EDTA) buffer at a temperature of 42xc2x0 C. and remaining bound when subject to washing at 42xc2x0 C. with 0.2xc3x97SSPE; preferably hybridizing in a buffer comprising 50% formamide in 5xc3x97SSPE buffer at a temperature of 42xc2x0 C. and remaining bound when subject to washing at 42xc2x0 C. with 0.2 x SSPE buffer at 42xc2x0 C. R5.24 nucleic acids can also be distinguished using alignment algorithms, such as BLASTX (Altschul et al. (1990) Basic Local Alignment Search Tool, J Mol Biol 215, 403-410). In addition, the invention provides nucleic acids having a sequence about 60-70%, preferably about 70-80%, more preferably about 80-90%, more preferably about 90-95%, most preferably about 95-99% similar to SEQ ID NO:1 as determined by Best Fit analysis using default settings.
The subject nucleic acids are of synthetic/non-natural sequences and/or are recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome. The subject recombinant nucleic acids comprising the nucleotide sequence of disclosed vertebrate R5.24 nucleic acids, or fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by (i.e. contiguous with) a sequence other than that which it is joined to on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, more preferably fewer than 500 bp, which is at a terminus or is immediately flanked by a sequence other than that which it is joined to on a natural chromosome. While the nucleic acids are usually RNA or DNA, it is often advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability, etc.
The subject nucleic acids find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, etc.; use in detecting the presence of R5.24 genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional R5.24 homologs and structural analogs.